Inhibition of Alpha-Glucosidase and Antioxidant Test of Stem Bark Extracts of Garcinia fruticosa Lauterb

Introduction: Diabetes mellitus (DM) is one of the global health emergencies that characterized by high blood glucose levels (hyperglycemia). Type 2 DM is the most common type in diabetic populations. Inhibition of alphaglucosidase can ameliorate postprandial hyperglycemia that occurs in patients with type 2 DM. Adding antioxidants to the therapy of DM is intended to reduce complications caused by oxidative stress. Some species of Garcinia have been proven to inhibit alpha-glucosidase and have antioxidant activity, but there is no research on Garcinia fruticosa Lauterb. Therefore, the aims of this research were to determine the activity of Garcinia fruticosa Lauterb. stem bark in inhibiting alpha-glucosidase and as an antioxidant. Methods: In this research, the Garcinia fruticosa Lauterb. stem bark was dried, grinded, and extracted by multistage maceration using n-hexane, ethyl acetate, and methanol. Inhibition of alpha-glucosidase test has been done in vitro on concentrated extracts and measured by microplate reader at 400 nm. The antioxidant test has been done using DPPH scavenging method and was measured by microplate reader at 519 nm. Results: Ethyl acetate extract is the most active extract for both test. IC50 values for inhibition of alpha-glucosidase test are 20.18 μg/mL that is more active than standard (acarbose) which has IC50 value 141.55 μg/mL. Meanwhile, IC50 value from an antioxidant test is 8.93 μg/mL that is not more active than standard (quercetin) which has IC50 value 2.51 μg/mL. Conclusion: Phytochemical screening shows that the ethyl acetate extract contains alkaloids, flavonoids, glycosides, saponins, and tannins.


INTRODUCTION
Diabetes mellitus (DM) is one of the leading causes of death and disability in worldwide. 1DM is characterized by hyperglycemia caused by abnormalities in insulin secretion, insulin action, or both.Prevalence of DM increase annually. 2Inhibition of alpha-glucosidase enzyme is one of the antidiabetic mechanism that uses in DM therapy, for example, acarbose.This mechanism can inhibit glucose absorption so can prevent hyperglycemia. 3 As a single therapy, acarbose is less effective because it is only 2% absorbed. 4hronic hyperglycemia can cause many complications such as damage, dysfunction and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. 1 Hyperglycemia has an important contribution in causing complications because of the trigger in free radicals reaction. 5Adding antioxidants in the DM therapy may prevent oxidative stress that caused by free radicals so that complications can be prevented. 6here are many kinds of research on species of Garcinia about their activity as alpha-glucosidase inhibitors and antioxidants but there is no research on Garcinia fruticosa Lauterb.Ethanolic extract of G. daedalanthera Pierre.stem barks showed that the extract can inhibit α-glucosidase with IC 50 value 3.71 µg/ml. 7Besides that, methanolic extract of G. lateriflora Blume varJavanica Boerlleaves showed that the extract has antioxidant activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method with IC 50 value 6.18 µg/ml. 8Based on chemotaxonomic consideration, Garcinia fruticosa Lauterb.could be expected to inhibit alpha-glucosidase and has antioxidant activity so could be used as a DM therapy.

Plant Material
The stem bark of Garcinia fruticosa Lauterb.was collected in January 2016 from Bogor, Indonesia and identified by Center for Plant Conservation-Bogor Botanical Garden.

Extraction
The dried stem bark of Garcinia fruticosa Lauterb.(780 g) was powdered and extracted consecutively with n-hexane, ethyl acetate, and methanol by cold maceration and then evaporated.On each extract is performed inhibition of alpha-glucosidase test and antioxidant test using DPPH scavenging method.

Inhibition of Alpha-Glucosidase Test
Pharmacognosy Journal, Vol 9, Issue 2, Mar-Apr, 2017 µg/mL; IC 50 value for ethyl acetate extract is 20.18µg/mL; and IC 50 value for methanol extract is 48.88 µg/mL (Table 1).IC 50 value ethyl acetate and methanol extract lower than acarbose.That means ethyl acetate and methanol extract is better in inhibiting alpha-glucosidase than acarbose.The ethyl acetate extract is the most active extract in this test because it has the lowest IC 50 value.This result is related to chemical compounds in the extract that can inhibit alpha-glucosidase synergistically, in contrast to acarbose which is a single compound.

Antioxidant Activity Test
The antioxidant test was performed using DPPH scavenging method by microplate reader (Versamax ELISA Microplate Reader, USA) at 519 nm that was the maximum wavelength of DPPH.Quercetin is used as a standard.The result of the antioxidant test for quercetin showed that quercetin has antioxidant activity with IC 50 2.51 µg/mL.Preliminary antioxidant test for extracts was done using n-hexane extract, ethyl acetate extract, and methanol extract with same concentration (100 µg/ mL).The n-Hexane extract has percent DPPH quenched 17.53%, ethyl acetate extract has 46,52%, and methanol extract has 35,98% (Table 2).Therefore, the most active extract in having antioxidant activity is ethyl acetate extract because it has the highest percent DPPH quenched.This extract was performed to determine IC 50 value.The result shows that the ethyl acetate extract has IC 50 8.93 µg/mL.IC 50 value extract is higher than standard (quercetin).In the other words, the extract is not more active than quercetin.However, based on Blois classification the extract is a very strong antioxidant because of the IC 50 value lower than 50 µg/mL.The strong antioxidant activity is related to phenolic and flavonoid compounds contained in extracts. 11

Phytochemical Screening
The screening was done on the most active extract in both inhibitions of alpha-glucosidase test and antioxidant activity test that is ethyl acetate extract.The results from phytochemical screening show that the extract contains alkaloids, flavonoids, glycosides, tannins, and saponins (Table 3).Alkaloids are discovered can inhibit the alpha-glucosidase activity competitively and non-competitively. 12Flavonoids can inhibit alpha-glucosidase activity and have antioxidant activity because of the hydroxyl groups. 13,14Glycosides also have a role in inhibiting alpha-glucosidase because of the similar structure substrate (contains glucose) so that glycosides can bind to active site. 7Tannins have a role in inhibiting alphaglucosidase because those can bind to protein make complexes. 15The hydroxyl groups in tannins have roles in inhibiting alpha-glucosidase and antioxidant activity. 8,12e inhibition of alpha-glucosidase was determined using adopted method. 9Five mg (equivalent to 90.3 units alpha-glucosidase enzyme) of alpha-glucosidase (Saccharomyces cerevisiae, Sigma-Aldrich, Germany) was dissolved in 50,0mL of phosphate buffer (pH 6.8) containing 100 mg of bovine serum albumin (Sigma-Aldrich, USA) and then diluted 152 µL in 5,0 mL with phosphate buffer (pH 6.8).The reaction mixture consisting 30 μL of samples at varying concentrations was premixed with 36μLphosphate buffer pH 6.8 and 17 μL of 5 mMp-nitrophenylα-D-glucopyranoside (Sigma-Aldrich, Switzerland).After preincubating at 39 o C for 5 min, 17 μ Lalpha-glucosidase(0.045 units/mL) was added and incubated at 39 o C for 15 minutes.The reaction was terminated by adding100μLNa 2 CO 3 200 mM.Inhibition of alpha-glucosidase was determined at 400 nm using microplate reader (Versamax ELISA Microplate Reader, USA) by measuring the quantity of p-nitrophenol released from p-NPG.Acarbose was used as positive control of α-glucosidase inhibitor.The concentration of the extract required to inhibit 50% of α-glucosidase activity under the assay conditions was defined as the IC 50 value.

Antioxidant Activity Test
The DPPH scavenging method was adopted from Bobo-Garcia et al. 10 The preliminary antioxidant test was done using n-hexane extract, ethyl acetate extract, and methanol extract with same concentration (100 µg/ mL).The IC 50 value was determined on the most active extract.The reaction mixture consisting 20 μL of diluted samples at varying concentrations was added to 180 μL of DPPH solution (150 μmol/L) in methanolwater (80:20, v/v) and shaken for 60 seconds in a 96-well microplate.After 40 minutes in the dark at room temperature, the absorbance was measured at 519 nm in the microplate reader of Versamax ELISA Microplate Reader (USA).Quercetin was used as a standard at 1.5-3.5 μg/mL.The % DPPH quenched was calculated using: where A sample is the absorbance at 519 nm of 20 μL of extract or standard with 180 μL DPPH solution after 40min; A blank is an absorbance at 519 nm of 20 μL of water with 180 μL methanol-water (80:20, v/v) after 40 min, and Controls the absorbance at 519 nm of 20 μL of water with 180 μL DPPH solution after 40 min.

Inhibition of Alpha-Glucosidase Test
Inhibition of alpha-glucosidase test was performed in optimal conditions for the enzyme that have been optimized.The optimal conditions include pH 6.8, temperature 39 o C, enzyme concentration 0.045 U/ mL, and substrate concentration 5 mM.This test use microplate reader (Versamax ELISA Microplate Reader) at 400 nm.Acarbose is used as a standard.The result shows that acarbose has high IC50 value 141.55 µg/ mL.This test was performed on all of the extracts with various concentrations.The results show that IC 50 value for n-hexane extract is 643.20